Page 1 of 73 Diabetes Glucagon-like peptide-1 receptor agonists increase pancreatic mass by induction of protein synthesis Jacqueline A. Koehler1, Laurie L. Baggio1, Xiemin Cao1, Tahmid Abdulla1, Jonathan E. Campbell, Thomas Secher2, Jacob Jelsing2, Brett Larsen1, Daniel J. Drucker1 From the1 Department of Medicine, Tanenbaum-Lunenfeld Research Institute, Mt. Sinai Hospital and 2Gubra, Hørsholm, Denmark Running title: GLP-1 increases pancreatic protein synthesis Key Words: glucagon-like peptide 1, glucagon-like peptide-1 receptor, incretin, exocrine pancreas Word Count 4,000 Figures 4 Tables 1 Address correspondence to: Daniel J. Drucker M.D. Lunenfeld-Tanenbaum Research Institute Mt. Sinai Hospital 600 University Ave TCP5-1004 Toronto Ontario Canada M5G 1X5 416-361-2661 V 416-361-2669 F [email protected] 1 Diabetes Publish Ahead of Print, published online October 2, 2014 Diabetes Page 2 of 73 Abstract Glucagon-like peptide-1 (GLP-1) controls glucose homeostasis by regulating secretion of insulin and glucagon through a single GLP-1 receptor (GLP-1R). GLP-1R agonists also increase pancreatic weight in some preclinical studies through poorly understood mechanisms. Here we demonstrate that the increase in pancreatic weight following activation of GLP-1R signaling in mice reflects an increase in acinar cell mass, without changes in ductal compartments or β-cell mass. GLP-1R agonists did not increase pancreatic DNA content or the number of Ki67+ cells in the exocrine compartment, however pancreatic protein content was increased in mice treated with exendin-4 or liraglutide. The increased pancreatic mass and protein content was independent of cholecystokinin receptors, associated with a rapid increase in S6 kinase phosphorylation, and mediated through the GLP-1 receptor. Rapamycin abrogated the GLP-1R- dependent increase in pancreatic mass but had no effect on the robust induction of Reg3α and Reg3β gene expression. Mass spectrometry analysis identified GLP-1R-dependent up-regulation of proteins important for translation, and export, including cytochrome P450, Fam129a, eIF4a1, Wars, and Dmbt1. Hence, pharmacological GLP-1R activation induces protein synthesis leading to increased pancreatic mass independent of changes in DNA content or cell proliferation in mice. 2 Page 3 of 73 Diabetes Gut hormones secreted from specialized endocrine cells subserve multiple functions integrating control of food ingestion, gut motility, and the digestion, absorption and assimilation of nutrients. The actions of enteroendocrine peptides to control lipid metabolism, body weight and glucose homeostasis have engendered considerable translational interest given the increasing incidence of dyslipidemia, obesity and diabetes. Glucagon-like peptide-1 (GLP-1), secreted from enteroendocrine L cells, reduces food intake, inhibits gastric emptying, and produces weight loss. GLP-1 also inhibits chylomicron secretion from enterocytes and lowers triglyceride levels in both preclinical and clinical studies (1). The most extensively studied action of GLP-1 is that of an incretin hormone, augmenting insulin and inhibiting glucagon secretion following meal ingestion, through actions targeting endocrine cells in the pancreas. Collectively, the glucoregulatory actions of incretin hormones led to development of two distinct drug classes that lower glucose by potentiation of incretin action, dipeptidyl peptidase-4 (DPP-4) inhibitors, and GLP-1 receptor (GLP-1R) agonists (2). Although classical glucoregulatory actions of incretin-based therapies target the endocrine pancreas, the non-glycemic actions of GLP-1R agonists and DPP-4 inhibitors on the exocrine pancreas have received considerable attention (3). Spontaneous reports of pancreatitis in diabetic patients treated with incretin-based therapies stimulated interest in whether GLP-1R agonists affect the exocrine pancreas (4). Although results of preclinical studies are conflicting, the majority of experiments do not link activation of GLP-1R signaling to enhanced susceptibility to pancreatitis in rodents (4; 5). Furthermore studies of transgenic reporter gene expression under the control of the endogenous murine Glp1r promoter (6), have not demonstrated GLP-1R expression in pancreatic acinar cells. 3 Diabetes Page 4 of 73 Despite lack of evidence for GLP-1R expression in acinar cells of the rodent pancreas, several preclinical studies have demonstrated that GLP-1R agonists increase the mass of the pancreas, predominantly in mice (7; 8) and in a subset of male non-human primates (9). Nevertheless, the increase in pancreatic weight following treatment with GLP-1R agonists has not been associated with histological abnormalities in the pancreas (3; 9; 10), and mechanistic explanations for changes in pancreatic mass have not been forthcoming. We show here that exendin-4 and liraglutide increase pancreatic weight via induction of protein synthesis, without changes in acinar cell proliferation or DNA content. These actions were independent of receptors for cholecystokinin, required the classical Glp1r, and were abrogated by inhibition of the mammalian target of rapamycin (mTor). Our findings provide an explanation for changes in pancreatic mass observed following treatment with GLP-1R agonists. 4 Page 5 of 73 Diabetes RESEARCH DESIGN AND METHODS Reagents, Animals and Treatments: Exendin-4 (Ex-4) was from Chi Scientific (Maynard, MA), liraglutide was from Novo Nordisk (Bagsværd, Denmark), rapamycin (Rapamune) was from Wyeth (Montreal, Quebec). Peptides were dissolved in phosphate buffered saline (PBS, vehicle) and administered to mice by intraperitoneal (ip) injection (Ex-4;10 nmol/kg, BID, or liraglutide;75 µg/kg, BID). Rapamycin was resuspended in 0.5% carboxymethacellulose (Sigma, C4888), 2.5% tween-80. C57BL/6J, Cckar-/-, and Cckbr-/- mice were from The Jackson Laboratory (Bar Harbor, ME). Whole body Glp1r−/− mice in the C57BL/6 background (11; 12) and wildtype (WT) littermate control mice were generated by crossing Glp1r+/− mice. Cckar-/-:Cckbr-/- (DKO) and WT littermate control mice were generated by crossing Cckar+/- and Cckbr+/- mice. Animal experiments were approved by the Animal Care Committee of the Mount Sinai Hospital. Pancreatic growth: Male C57BL/6 mice (8-10 weeks-old) were administered exogenous Ex-4 or vehicle (PBS) for 7 days or 4 weeks. Male (not shown) and female Cckbr-/-, Cckar-/-, (7- 12 weeks-old) and Cckar:Cckbr-/- (DKO) (8-13 weeks-old) and WT littermate control mice were administered exogenous Ex-4 or vehicle for 10 days. Non-fasted mice were euthanized by CO2 inhalation in the morning ~12h after the last injection) unless otherwise indicated. Time course: male C57BL/6 mice (11 weeks-old) were injected with Ex-4 or vehicle every 12h and pancreata obtained at 4 h, 12 h or 24h-7 days. For the 24h-7 day time points, mice were euthanized in the morning ~12 h after the last injection). Rapamycin study: male C57BL/6 mice (8-10 weeks-old) were administered vehicle (0.5% carboxymethacellulose, 2.5% tween-80) or rapamycin (2 mg/kg 1x daily ip) 30 min prior to the first (morning) injection of exogenous Ex-4, or PBS for 3 or 7 days. High protein diet (HPD) study: HPD (AIN-93M modified to contain 75% Casein, 5 Diabetes Page 6 of 73 D1206504) and control diet (CD) AIN-93M (D10012M) were from Research Diets (New Brunswick, N.J.). Glp1r−/− and WT littermate control female mice (8-11 weeks old) were acclimatized to the CD for 1 week prior to being fed the HPD or CD ad libitum for 7 days. Tissue collection, immunohistochemistry, DNA/protein and water content: Following euthanasia, mice were weighed, blood samples collected by cardiac puncture, and serum was stored at -80°C. The pancreas was removed, weighed and cut in half lengthwise; one-half was fixed in 10% formalin for 24h, the other half was cut into 4 equal sections from tail (attached to spleen) to head for RNA, protein, DNA/protein content, and water content. Immunohistochemistry and morphometry were done on 5-µm histological sections stained with hematoxylin and eosin (H&E) or Ki67 (Thermo Scientific, RM9106-S1, 1:2000) by standard procedures. Sections were scanned and analyzed using the Scanscope CS system (Aperio Technologies) at x20 magnification. Immunohistochemistry for phospho-S6 ribosomal protein (Cell Signaling, Ser 240/244, D68F8 XP) was performed according to manufacturer’s instructions. Immunohistochemistry for Reg3 (antisera, 1:200 dilution, Dr. Rolf Graf, University Hospital Zurich, Zurich, Switzerland) was performed using sodium citrate buffer pH 6.0 for antigen unmasking and overnight incubation with the Reg3 antibody. Edema was calculated following desiccation for 72 h and expressed as a percentage of wet weight (wet weight-dry weight/wet weight x100). For analysis of DNA and protein content, pancreas samples were weighed, homogenized in a lysis buffer containing 0.1% Tx-100, 5mM MgCl2, and sonicated for 15 sec. Protein content was measured using Bradford assay (Bio-Rad) and a Pierce BCA protein assay kit (Thermo Scientific) and DNA content was measured using a DNA quantification kit (Sigma, DNAQF). Serum amylase and lipase levels: 6 Page 7 of 73 Diabetes Serum amylase activity was measured using the Phadebas amylase test (Magle Life Sciences, Cambridge, MA) and serum lipase activity was analyzed with the Lipase color assay (905-B, Sekisui Diagnostics, Charlottetown, PE). Serum amylase and lipase activity levels were also measured in serum samples from mice with secretagogue-induced pancreatitis induced by administration of five sequential